PROJECT SUMMARY Malaria is one of the world?s major public health threats. The disease is transmitted when an infected female Anopheles mosquito, while probing for a blood meal, injects saliva together with Plasmodium sporozoites into the skin of the vertebrate host (1, 2). To date, a highly effective, safe and FDA-approved human vaccine against malaria has not been developed. The most established malaria vaccine candidate, RTS,S/AS01, which is a recombinant protein based on the circumsporozoite protein of Plasmodium falciparum, confers moderate protection against malaria which wanes over time (1-10). There is great need for an improved vaccine against malaria, and that is the goal of this proposal - using a protein in mosquito saliva that influences the ability of Plasmodium sporozoites to infect the vertebrate host. In our recently published study (11), we identified a protein in the saliva of female Anopheles gambiae mosquitoes, named AgTRIO. We produced recombinant AgTRIO in Escherichia coli and demonstrated that active immunization of mice with recombinant AgTRIO significantly reduced mosquito-borne Plasmodium berghei and Plasmodium falciparum infection of the animals. This work showed that a protein in mosquito saliva can be targeted to prevent Plasmodium infection. The aim of this proposal is to develop a strategy for active immunization with AgTRIO protein produced in a eukaryotic expression system. Our long-term goal is to develop a vaccine that will provide robust protection against malaria for use in humans.